A fundamental feature of mammalian spermatogenesis is the continual production of spermatozoa within the testis throughout the reproductive lifetime of the animal. It takes many weeks for a progenitor cell to become a functional sperm yet it has been estimated that the human testis produces 1000 sperm with each heartbeat or about 37 billion sperm per year. To achieve and sustain this immense level of production, the pool of progenitor cells and the commitment of cells to differentiation must be carefully coordinated and maintained. This proposal addresses fundamental unanswered questions relating to how the commitment of undifferentiated A spermatogonia (SpA) to differentiation, and ultimately to meiosis, is both initiated and maintained. Retinoic acid (RA) is essential for spermatogonial differentiation yet little is known about how it is generated and controlled within the testis. This proposal will test the hypothesis that RA concentrations are tightly regulated by RDH10 in Sertoli cells and degraded by CYP26A1 in germ cells. This proposal will also test the hypothesis that the periodic differentiation of spermatogonia in the juvenile and adult testis is due to the appearance of preleptotene spermatocytes that trigger an increase in RA at different points along the length of the tubule. Specific Aim 1 will determine whether RDH10 in Sertoli cells and CYP26A1 in germ cells are required for the initial differentiation of SpA. This aim will also investigate whether patches of the tubules containing high levels of RA activity colocalize with markers of differentiating spermatogonia and whether the spermatogonial stem cell population tends to reside in areas of the tubule with low levels of RA. Specific Aim 2 will investigate how spermatogonial differentiation occurs in a periodic manner in the juvenile testis by utilizing an innovative technique that allows for the first wave o spermatogenesis to be synchronized. By synchronizing the first wave, we will, for the first time, be able to accurately measure changes in the levels of RA as spermatogonia differentiate to become spermatocytes, investigate whether RDH10 and CYP26A1 are required for cyclic increases in RA concentration, and determine whether the preleptotene spermatocytes can trigger these increases in RA. Specific Aim 3 will investigate whether other members of the retinoid metabolizing and signaling family display cell-specific expression profiles as the spermatogonia differentiate as well as profile global changes in the translatome of these cells. In addition, this aim will identify specific markers of the different types of differentiating spermatogonia, filling a gap in our current understanding of these cell types. Collectively, these data will enhance the current understanding of how spermatogonia differentiate and commit to undergoing meiosis and describe, for the first time, a mechanism for how the asynchronous production of sperm is not only initiated but maintained.